Ionomic Parameters of Populations of Common Juniper (Juniperus communis L.) Depending on the Habitat Type

For the study of the ionomic parameters of Juniperus communis needles, fourteen sites covering most of the territory of Lithuania and belonging to distinct habitats (coastal brown dunes covered with natural Scots pine forests (G), Juniperus communis scrubs (F), transition mires and quaking bogs (D), subcontinental moss Scots pine forests (G), and xero-thermophile fringes) were selected. Concentrations of macro-, micro-, and non-essential elements were analyzed in current-year needles, sampled in September. According to the concentrations of elements in J. communis needles, the differences between the most contrasting populations were as follows: up to 2-fold for Mg, N, K, Ca, and Zn; 2- to 7-fold for P, Na, Fe, Cu, Al, Cr, Ni, and Pb; and 26- to 31-fold for Mn and Cd. The concentrations of Cd, Cr, and Ni in needles of J. communis did not reach levels harmful for conifers. When compared to all other habitats (B, F, G, and E), the populations from transition mires and quaking bogs (D) had significantly lower concentrations of main nutritional elements N (12176 µg/g d. m.), P (1054 µg/g d. m.), and K (2916 µg/g d. m.). In Juniperus communis scrubs (F), a habitat protected by EUNIS, the concentration of K in the needles was highest, while Zn and Cu concentrations were the lowest. Principal component (PC) analyses using concentrations of 15 elements as variables for the discrimination of populations or habitats allowed authors to distinguish F and B habitats from the E habitat (PC1) and F and D habitats from the G habitat (PC2). Discriminating between populations, the most important variables were concentrations of P, N, Mg, Ca, Cu, and K. Discriminating between habitats, the important variables were concentrations of N and P.


Introduction
Common juniper (Juniperus communis L.) is a coniferous tree or shrub belonging to the Cupressaceae family. It is one of the most widespread plant species in the world [1]. Juniperus communis grows in the northern hemisphere [2]. Its distribution range encompasses four continents, with a European range from Ireland to Russia in longitude and from Spain to Scandinavia in latitude [3]. Although J. communis is the most widespread conifer in the world, it has become an endangered species in some European locations [4]. An increased    The concentrations of microelements (Mn, Fe, Na, Zn, Cu) in the needles of J. communis populations ranged from 30.2 to 939.2 µg/g d. m. for Mn (with lowest concentration in the population 10 E, and the highest concentration in the 4 G), from 20.2 to 115 µg/g d. m. for Fe (with the lowest concentration in the 9 E, and the highest in the 1 B), from 2.84 to 9.49 µg/g d. m. for Na (with the lowest concentration in the 12 G, and the highest in the 1 B), from 12.58 to 22.42 µg/g d. m. for Zn (with the lowest concentration in the 11 E, and the highest in the population 7 G), and from 1.10 to 7.46 µg/g d. m. for Cu (with the lowest concentration in the 8 D, the highest in the 12 G).
The concentrations of non-essential elements (Al, Ni, Pb, Cr, Cd) in the needles of J. communis ranged from 14. 35  The comparison of populations according to habitat type ( Figure 2) showed that the concentration of N in the needles of J. communis were 1.14 to 1.37 times lower (p < 0.05) in the transition mires and quaking bogs (D) when compared to other habitats (coastal brown dunes covered with natural Scots pine forests (G), Juniperus communis scrubs (F), subcontinental moss Scots pine forests (G), and xero-thermophile fringes (E)). Needle concentration of N was 1.20 to 1.37 times higher (p < 0.05) in the subcontinental moss Scots pine forests (G) compared to the coastal brown dunes covered with natural Scots pine forests (B), J. communis scrubs (F), and transition mires and quaking bogs (D).
The concentration of Ca in the needles of J. communis was 1.27 to 1.31 times lower (p < 0.05) in Juniperus communis scrubs (F) than in coastal brown dunes covered with natural Scots pine forests (B) and transition mires and quaking bogs (D). In the transition mires and quaking bogs (D), Ca concentration was 1.20 to 1.53 times higher (p < 0.05) when compared to coastal brown dunes covered with natural Scots pine forests (B), Juniperus communis scrubs (F), and subcontinental moss Scots pine forests (G). In coastal brown dunes covered with natural Scots pine forests (B), the concentration of Ca was 1.20 to 1.27 times higher (p < 0.05) than that in Juniperus communis scrubs (F) and subcontinental moss Scots pine forests (G). In xero-thermophile fringes (E), the concentration of Ca was 1.19 to 1.31 times higher (p < 0.05) than that in Juniperus communis scrubs (F) and subcontinental moss Scots pine forests (G). The concentration of P in the needles of J. communis was 1.42 to 1.56 times lower (p < 0.05) in subcontinental moss Scots pine forests (G) when compared to Juniperus communis scrubs (F) and transition mires and quaking bogs (D), 1.52 to 1.67 times higher (p < 0.05) in subcontinental moss Scots pine forests (G) than in Juniperus communis scrubs (F) and transition mires and quaking bogs (D), and 1.36 times (p < 0.05) higher in xerothermophile fringes (E) when compared to transition mires and quaking bogs (D). The concentration of K in the needles of J. communis was 1.12 to 1.13 times lower (p < 0.05) in transition mires and quaking bogs (D) than in coastal brown dunes covered with natural Scots pine forests (B), Juniperus communis scrubs (F), and subcontinental moss Scots pine forests (G), and 1.08 times lower (p < 0.05) in xero-thermophile fringes (E) than in Juniperus communis scrubs (F). The concentration of Mg in the needles of J. communis was 1.02 to 1.04 times higher (p < 0.05) in subcontinental moss Scots pine forests (G) when compared to coastal brown dunes covered with natural Scots pine forests (B), Juniperus communis scrubs (F), and transition mires and quaking bogs (D). The concentration of Ca in the needles of J. communis was 1.27 to 1.31 times lower (p < 0.05) in Juniperus communis scrubs (F) than in coastal brown dunes covered with natural Scots pine forests (B) and transition mires and quaking bogs (D). In the transition mires and quaking bogs (D), Ca concentration was 1.20 to 1.53 times higher (p < 0.05) when compared to coastal brown dunes covered with natural Scots pine forests (B), Juniperus communis scrubs (F), and subcontinental moss Scots pine forests (G). In coastal brown dunes covered with natural Scots pine forests (B), the concentration of Ca was 1.20 to 1.27 times higher (p < 0.05) than that in Juniperus communis scrubs (F) and subcontinental moss Scots pine forests (G). In xero-thermophile fringes (E), the concentration of Ca was 1.19 to 1.31 times higher (p < 0.05) than that in Juniperus communis scrubs (F) and subcontinental moss Scots pine forests (G). The concentration of P in the needles of J. communis was 1.42 to 1.56 times lower (p < 0.05) in subcontinental moss Scots pine forests (G) when compared to Juniperus communis scrubs (F) and transition mires and quaking bogs (D), 1.52 to 1.67 The concentration of Mn in the needles of J. communis was 1.23 to 10.45 times higher (p < 0.05) in coastal brown dunes covered with natural Scots pine forests (B) when compared to J. communis scrubs (F), transition mires and quaking bogs (D), and xero-thermophile fringes (E). In Juniperus communis scrubs (F) and subcontinental moss Scots pine forests (G), the concentration of Mn was 6.35 to 8.53 and 8.14 to 10.94 times higher (p < 0.05), respectively, when compared to transition mires and quaking bogs (D) and xero-thermophile fringes (E). The concentration of Fe in the needles of J. communis was 1.59 to 2.20 times higher (p < 0.05) in coastal brown dunes covered with natural Scots pine forests (B) than in transition mires and quaking bogs (D), subcontinental moss Scots pine forests (G), and xero-thermophile fringes (E). For the Juniperus communis scrubs (F) the concentration of Fe was 1.46 to 1.67 times higher (p < 0.05) when compared to subcontinental moss Scots pine forests (G) and xero-thermophile fringes (E). The concentration of Na in the needles of J. communis Plants 2023, 12, 961 6 of 19 was 1.24 to 2.00 times higher (p < 0.05) in coastal brown dunes covered with natural Scots pine forests (B) when compared to other habitats. For Juniperus communis scrubs (F), the concentration of Na was 1.43 to 1.61 times higher (p < 0.05) than that in subcontinental moss Scots pine forests (G) and xero-thermophile fringes (E). The concentration of Zn in the needles of J. communis was 1.45 to 1.31 times higher (p < 0.05) in Juniperus communis scrubs (F) when compared to xero-thermophile fringes (E). In subcontinental moss Scots pine forests (G) the concentration of Zn was 1.29 times higher (p < 0.05) than in xero-thermophile fringes (E). For the Juniperus communis scrubs (F), the concentration of Zn was 1.31 to 1.39 times lower (p < 0.05) when compared to that in transition mires and quaking bogs (D) and subcontinental moss Scots pine forests (G). The concentration of Cu in the needles of J. communis was 2.37 to 2.91 times higher (p < 0.05) in coastal brown dunes covered with natural Scots pine forests (B) when compared to Juniperus communis scrubs (F) and transition mires and quaking bogs (D). In subcontinental moss Scots pine forests (G) and xero-thermophile fringes (E) the concentration of Cu was 2.63 to 3.23 and 2.94 to 3.62 times higher (p < 0.05), respectively, than that in transition mires and quaking bogs (D) and xero-thermophile fringes (E).
The concentration of Al in the needles of J. communis was 1.42 to 2.14 times lower (p < 0.05) in coastal brown dunes covered with natural Scots pine forests (B) when compared to other habitats. For Juniperus communis scrubs (F), the concentration of Al was 1.39 to 2.09 times lower (p < 0.05) when compared to other habitats. In transition mires and quaking bogs (D), the concentration was 1.32 to 1.51 times lower (p < 0.05) than that in subcontinental moss Scots pine forests (G) and xero-thermophile fringes (E). The concentration of Ni in the needles of J. communis was 1.65 to 7.10 times higher (p < 0.05) in coastal brown dunes covered with natural Scots pine forests (B) when compared to other habitats. In subcontinental moss Scots pine forests (G), the concentration of Ni was 2.47 higher (p < 0.05) than in Juniperus communis scrubs (F). In transition mires and quaking bogs (D), the concentration of Ni was 3.20 to 4.30 times lower (p < 0.05) than in coastal brown dunes covered with natural Scots pine forests (B), Juniperus communis scrubs (F), subcontinental moss Scots pine forests (G), and xero-thermophile fringes (E). The concentration of Pb in the needles of J. communis was 1.50 to 2.18 times lower (p < 0.05) in subcontinental moss Scots pine forests (G) when compared to Juniperus communis scrubs (F) and xero-thermophile fringes (E). The concentration of Cr in the needles of J. communis was 1.19 to 1.92 times higher (p < 0.05) in coastal brown dunes covered with natural Scots pine forests (B) when compared to other habitats. For Juniperus communis scrubs (F), the concentration of Cr was 1.16 to 1.19 times higher (p < 0.05) than in transition mires and quaking bogs (D) and subcontinental moss Scots pine forests (G). The concentration of Cd in the needles of J. communis was 2.26 to 9.51 times higher (p < 0.05) in coastal brown dunes covered with natural Scots pine forests (B) when compared to other habitats. For Juniperus communis scrubs (F), the concentration of Cd was 3.21 to 3.67 times higher (p < 0.05) when compared to that in transition mires and quaking bogs (D) and xero-thermophile fringes (E). In subcontinental moss Scots pine forests (G), the concentration was 2.80 to 3.67 times higher (p < 0.05) than that in transition mires and quaking bogs (D) and xero-thermophile fringes (E).
To relate concentrations of macro-, micro-, and non-essential elements of J. communis populations, Pearson rank correlations were estimated ( Figure 3). The concentration of N was positively correlated with the concentrations of P (Rs = 0.79, p < 0.001) and Mg (Rs = 0.60, p < 0.0054) and negatively correlated with Ca (Rs = -0.54, p < 0.020). The concentration of P was positively correlated with the concentration of K (Rs = 0.55, p < 0.015) and negatively correlated with Ca (Rs = -0.60, p < 0.002). The concentration of Mg was positively correlated with the concentration of Al (Rs = 0.71, p < 0.001) and Cu (Rs = 0.53, p < 0.032) and negatively with Cr (Rs = -0.52, p < 0.042). The concentration of Fe was positively correlated with concentrations of Cr (Rs = 0.57, p < 0.009) and Cd (Rs = 0.55, p < 0.016). The concentration of Mn was positively correlated with the concentration of Cd (Rs = 0.57, p < 0.007). The concentration of Al was positively correlated with the concentration of Cu (Rs = 0.62, p < 0.01) and negatively correlated with Cd (Rs = -0.52, p < 0.035). The concentration of Na was positively correlated with the concentration of Pb (Rs = 0.57, p < 0.007) and Cr (Rs = 0.56, p < 0.010). The concentration of Cr was positively correlated with the concentration of Cd (Rs = 0.60, p < 0.003). The concentration of Ni was positively correlated with the concentration of Cd (Rs = 0.58, p < 0.006). Concentration of K, Ca, Zn, Cu, and Pb did not correlate with concentration of any other element. 0.016). The concentration of Mn was positively correlated with the con (Rs = 0.57, p < 0.007). The concentration of Al was positively corr concentration of Cu (Rs = 0.62, p < 0.01) and negatively correlated with C 0.035). The concentration of Na was positively correlated with the concen = 0.57, p < 0.007) and Cr (Rs = 0.56, p < 0.010). The concentration of C correlated with the concentration of Cd (Rs = 0.60, p < 0.003). The concent positively correlated with the concentration of Cd (Rs = 0.58, p < 0.006). C K, Ca, Zn, Cu, and Pb did not correlate with concentration of any other e  To explain the most important macroelements, microelements, and non-essential elements on the variability of J. communis populations, principal component (PC) analysis was performed ( Figure 4). The first four PCs were highly informative and together accounted for 79.8% of the overall variance. Individually, PC1, PC2, PC3, and PC4 accounted for 29.2% (4.071), 27.1% (3.846), 15.2% (2.103), and 8.3% (1.262) of variance, respectively. Contribution of most elements to PC1 was positive and that of N, Ca, Cu, Mg, and Al was negative. The contribution of most elements to PC2 was positive, and it was negative in case of Fe, Cr, Na, Pb, and Ca. According to the variability of elemental concentrations, displayed in the biplot of two PC components, the importance of variables (in descending order) in PC1 was as follows: Cd > Al > Cr > Fe > Mg > Ni > Zn > K > Mn > Na > Cu > Pb > P > Ca > N. The order of importance of variables in PC2 was different: P > N > Mg > Ca > Cu > K > Pb > Mn > Al > Ni > Zn > Cd > Na > Cr > Fe.
(in descending order) in PC1 was as follows: Cd > Al > Cr > Fe > Mg Na > Cu > Pb > P > Ca > N. The order of importance of variables in N > Mg > Ca > Cu > K > Pb > Mn > Al > Ni > Zn > Cd > Na > Cr > Fe  According to the variability of elemental concentrations, displayed in the biplot of two PC, the importance of variables (in descending order) in PC1 was as follows: Fe > Cd > Na > Cr > Al > Mg > Mn > K > Ni > Pb > Zn > Ca > P > N > Cu. The order of importance of variables in PC2 was different: N > P > Cu > Pb > Ni > Mg > Mn > Al > K > Ca > Zn > Cd > Fe > Cr > Na.
In Lithuania, the ionome of Pinus sylvestris and Picea abies were exam industrial pollution sources [55,56]. The only assessment of Juniperus co was performed two decades earlier, regardless of the needles' age [40].

Macroelements
In conifer needles, a concentration of N lower than 12,000-14,000 considered a nitrogen deficiency. Normal or ecological optimum values 14,000 and 18,000 µg/g d. m. Ecological excess or physiological optimum 18,000 to 23,500 µg g -1 d. m., and physiological excess values that affect growth of the plant are from 23,500 to 35,000 µg/g d. m. [48,[50][51][52]. In communis, the mean concentration of N in current-year needles (Figure 1)
In Lithuania, the ionome of Pinus sylvestris and Picea abies were examined near local industrial pollution sources [55,56]. The only assessment of Juniperus communis ionome was performed two decades earlier, regardless of the needles' age [40].

Macroelements
In conifer needles, a concentration of N lower than 12,000-14,000 µg/g d. m. is considered a nitrogen deficiency. Normal or ecological optimum values range between 14,000 and 18,000 µg/g d. m. Ecological excess or physiological optimum values are from 18,000 to 23,500 µg g -1 d. m., and physiological excess values that affect and inhibit the growth of the plant are from 23,500 to 35,000 µg/g d. m. [48,[50][51][52]. In our study of J. communis, the mean concentration of N in current-year needles (Figure 1) was 15,422 µg/g d. m. The mean concentration of N in same age needles of J. communis in Italy was much lower (8450 µg/g d. m.) [46]. The concentration of N in current-year needles of Scot pine in Lithuania (near industrial pollution sources) ranged from 12,300 to 20,030 µg/g d. m. [56]. The concentration of Ca in conifer needles lower than 400 to 2000 µg/g d. m. is considered a deficiency [48,55]. The mean concentration of Ca in J. communis in Lithuania was 10,352 µg/g d. m. and in Italy it was 12,300 µg/g d. m. [46]. The concentration of Ca in Scot pine needles in Lithuania ranged between 6100 and 10,400 µg/g d. m. [56]. In conifer needles, a concentration of P lower than 1300 µg g -1 d. m. is considered a phosphorus deficiency [48]. In our study, the mean concentration of P in J. communis was 1516 µg/g d. m.; this was similar to the concentration of P in Pinus sylvestris in Lithuania (980 to 1480 µg/g d. m.) [56] and Scandinavian countries (819 to 2120 µg/g d. m) [48]. In Italian Juniperus communis, the mean concentration of P was significantly higher (6720 µg/g d. m.) [46]. In conifer needles, a concentration of K lower than 4000 µg/g d. m. is considered a potassium deficiency [48]. The mean concentration of K in J. communis in Lithuania was 3145 µg/g d. m. and the mean concentration of K in J. communis in Italy was 3500 µg/g d. m. [46]. In Lithuanian Pinus sylvestris, the concentration of K ranged from 450 to 7650 µg/g d. m. [56]. In conifer needles, a concentration of lower than 250 to 400 µg/g d. m. is considered a major magnesium deficiency (visible symptoms can be detected) [57]. If the concentration is within 400 to 600 µg/g d. m., this is considered a minor deficiency (growth retardation might be observed). Meanwhile, normal values range between 600 to 900 µg/g d. m. and optimal values are 800 to 1300 µg/g d. m. [46][47][48]. In our study, the mean concentration of Mg in J. communis was 608 µg/g d. m. The mean concentration of Mg in J. communis in Italy was three times higher (1800 µg/g d. m.) [38]; in Lithuanian P. sylvestris, the concentration of Mg ranged from 1200 to 1710 µg/g d. m. [56].

Microelements
In conifer needles, a concentration of Mn lower than 10 to 20 µg/g d. m. is considered a manganese deficiency [48]. In our study, the mean concentration of Mn in current-year needles of J. communis (Figure 1) was 275.4 µg/g d. m. The mean concentration of Mn in J. communis in Italy was much lower (85 µg/g d. m.) [46]. In contrast, the study conducted in Lithuania 20 years earlier reported the concentrations of Mn between the range of 177 to 2930 µg/g d. m. for J. communis and between the range of 33 to 3500 µg/g d. m. for Picea abies [40]. In conifer needles, a concentration of Fe higher than 500 µg/g d. m. is considered as ecological excess or physiological optimum values [48]. In our study, the mean concentration of Fe in J. communis was 65.5 µg/g d. m.; this was very similar to Italian data (54 µg/g d. m.) [46]. Iron concentration in Lithuanian Pinus sylvestris was in the range similar to that found in our study (44 to 94 µg/g d. m.) [56]. In conifer needles, a concentration of Zn lower than 8 to 15 µg/g d. m. is considered a deficiency in zinc; concentrations of 300 to 600 µg/g d. m. are poisonous to the plant [48]. In our study, the mean concentration of Zn in J. communis was 16.55 µg/g d. m. The concentration of Zn in the previous study (24 sites in Lithuania) ranged from 16 to 30 µg/g d. m [40], while the mean concentration of Zn in J. communis in Italy was 23 µg/g d. m. [46]. In the currentyear-four-year needles of Picea abies (48 sites in Lithuania) the concentration of Zn ranged from 10 to 72 µg/g d. m) [40], and the concentration of Zn in Lithuanian Pinus sylvestris was within the range of 42.9 to 75.3 µg/g d. m. [56]. In conifer needles, a concentration of Cu lower than 2 to 3 µg/g d. m. is considered a copper deficiency. Normal or ecological optimal values range between 5 and 30 µg/g d. m.; ecological excess or physiological optimum values range between 20 and 30 µg/g d. m., while concentrations between 20 and 100 µg/g d. m. are poisonous to the plant [48]. The mean concentration of Cu in J. communis in our study was 4.43 µg/g d. m., nearly the same as the mean concentration of Cu in J. communis in Italy (4 µg/g d. m.) [46] and within the range of concentrations of Cu in Pinus sylvestris in Lithuania (3.62 to 5.79 µg/g d. m.) [56]. Concentrations of K, Ca, Fe, Zn, Al, Cu, Ni, and Cd in our J. communis populations were very similar to Italian J. communis, growing quite distinct climates and edaphic conditions. This shows that nutrient concentrations are tightly related to genome. Such evidence further supports the validity of the Ellenberg indicatory value for soil richness along a wide geographical scale.

Non-Essential Elements
According to our study, the mean concentration of Al in the needles of J. communis was 25.26 µg/g d. m (Figure 1). The mean concentration of Al in J. communis in Italy was 30 µg/g d. m. [46] and concentrations of Al in Lithuanian Pinus sylvestris ranged from 25 to 190 µg/g d. m. [56]. In conifer needles, a concentration of Ni between 0.5 to 5 µg/g d. m. is considered as normal or ecologically optimal values, whereas concentrations of 10 to 100 µg/g d. m. are poisonous to the plant [39,58]. In our study, the mean concentration of Ni in J. communis was 0.73 µg/g d. m. The mean concentration of Ni in J. communis in Italy was 1 µg/g d. m. [46]. The concentrations of Ni in Lithuanian Pinus sylvestris ranged from 0.58 to 2.21 µg/g d. m. [56]. The mean concentration of Pb in J. communis in our study was 0.265 µg/g d. m. The concentrations of Pb in J. communis ranged from 0.19 to 1.23 µg/g s. m in the previous study in Lithuania [40]. The mean concentration of Pb in J. communis in Italy was several times higher (1 µg/g d. m.) when compared to our results [46]. The mean concentration of Pb in the current-year-four-year needles of P. abies in Lithuania ranged from 0.32 to 1.4 µg/g d. m. [40]; concentrations of Pb in Lithuanian Pinus sylvestris were within the range of 0.66 to 2.01 µg/g d. m. [56]. Our findings of low Pb concentrations in the needles of juniper corresponded to the background concentrations of this element found in the leaves of deciduous trees (Salix spp., Populus tremula L., Frangula alnus Mill., Quercus robur L., Tilia cordata Mill., Acer platanoides L., Alnus spp.), and herbaceous plants (Agrostis spp., Achillea millefolium L.) sampled in various sites in Lithuania [59]. In our study, the mean concentration of Cr in J. communis was 0.305 µg/g d. m. The concentration of Cr in J. communis in the previous Lithuanian study ranged between 0.07 and 0.39 µg/g s. m [40], the mean concentration of Cr in J. communis in Italy was 2 µg/g d. m. [46], and the concentration of Cr in the current-year-four-year needles in Norway spruce in Lithuania ranged from 0.12 to 0.45 µg/g d. m. [40]. The concentration of Fe and Ni in J. communis needles in Scotland (Fe 11.7-310 µg/g d. m.; Ni 6.9-32 µg/g d. m.) was similar to our results, but the concentration of Cr was higher in Scotland (Cr 9.7-15 µg/g d. m.) [58]. The mean concentration of Cd in J. communis in our study was 0.034 µg/g d. m. In the previous Lithuanian study, the concentrations of Cd in J. communis ranged from 0.03 to 0.23 µg/g d. m. [40], the mean concentration of Cd in J. communis in Italy was 0.01 µg/g d. m. [46], and the mean concentration of Cd in the current-year-four-year needles in Picea abies in Lithuania ranged from 0.01 to 0.29 µg/g d. m. [40].

Macro-, Micro-, Non-Essential Element Concentration Relationship to Habitat Type
Concentrations of nutrient elements in Juniperus thurifera needles varied widely and depended on habitat [60]. When compared to other habitats, J. communis growing in coastal brown dunes covered with natural Scots pine forests (B) had high needle concentrations of Fe, Zn, Ni, Na, Cr, Pb, and Cd, moderate concentrations of N, K, Ca, and Cu, and low concentrations of Mg and Al (Figure 2). The coastal brown dunes covered with natural Scots pine forests (B) are dominated by sandy soils covered by a thin layer of organic matter that is affected by intense wind erosion. Analysis of elements in J. communis needles showed that the concentration of Mg (591 µg/g d. m.) was significantly lower in coastal brown dunes covered with natural Scots pine forests (B) when compared to other habitats. The Mg concentration of 400 to 600 µg/g d. m. is considered as a mild deficiency [53,54]. Significantly higher concentrations of Na (9.49 µg/g d. m.) and Cr (0.531 µg/g d. m.) were also determined in the Scots pine forests habitat (B) when compared to other habitats (Juniperus communis scrubs (F), transition mires and quaking bogs (D), xero-thermophile fringes (E), and subcontinental moss Scots pine forests (G)). High concentrations of Na are typical for acidic or salty wetlands [61]. The high concentration Na found in population 1 B could be related to its proximity to the sea. Increased Na + ions may replace K + ions [62] and affect the plant's activity. The highest concentration of Cd was 0.105 µg/g d. m. In our study, concentrations of heavy metals (Cd, Cr, and Ni) in the needles of J. communis did not reach levels that are harmful for plants.
When compared to other habitats, the needles of J. communis growing in Juniperus communis scrubs (F) had high concentrations of K, moderate concentrations of Mn, and low concentrations of Zn and Cu. Juniperus communis scrubs (F) are distinguished by a high density of J. communis (1747 units/ha) [63]. Only the concentration of Zn (13.05 µg/g d. m.) was significantly lowest in Juniperus communis scrubs (F), within a range indicating a deficiency in conifer needles (8 to 15 µg/g d. m.) [64]. Zinc deficiency results in a stronger response when compared to the deficiency of other microelements [65]. Zinc influences the production of auxin, and when the production of this hormone is disrupted, plant growth might be limited [66,67].
According to studies in various European countries, ecological optimum of N in conifer needles is 14,000 to 18,000 µg/g d. m. and N deficiency is assumed in case its concentration is lower than 12,000 to 14,000 µg/g d. m. [48,68]. Juniperus communis needles of the same age sampled in the mixed oak-pine forest Querco-Pinetum (Poland) had very similar N concentration as in some habitats in our study (i.e., coastal brown dunes covered with natural Scots pine forests (B), Juniperus communis scrubs (F), and transition mires and quaking bogs (D)) and lower concentration than in the rest of our study habitats (i.e., subcontinental moss Scots pine forests (G) and xero-thermophile fringes (E)) [33].
Compared to all other habitats (B, F, G, and E), our populations in transition mires and quaking bogs (D) had significantly lower concentrations of the main nutritional elements N (12,176 µg/g d. m.), P (1054 µg/g d. m.), and K (2916 µg/g d. m.). Only the concentration of P in the populations in transition mires and quaking bogs (D) did not differ from the F habitat. When compared to other habitats, the mean concentrations in the needles in transition mires and quaking bogs (D) were highest for Ca, moderate for Fe, Zn, Al, and Pb, and lowest for N, P, K, Ni, Cr, and Cd. The concentrations of needle calcium causing a normal growth are 3000 to 12,800 µg/g d. m. and its deficiency falls into the interval of 400 to 3000 µg/g. m. [53]. In our study, needle deficiency in P (<1300 µg/g d. m. [48] was found in J. communis growing in transition mires and quaking bogs (D) and Juniperus communis scrubs (F). The highest concentration of Ca (14,723 µg/g d. m.) was found in population 8 D in the lakeside area, which is dominated by calcareous soil. Compared to other habitats, J. communis growing in transition mires and quaking bogs (D) had the lowest concentration of Ni (0.21 µg/g d. m.). Copper concentrations within a range of 5 to 30 µg/g d. m. are assumed as normal, whereas concentrations within 2 to 3 µg/g d. m. are deficient for conifer needles [48]. In our study, the lowest Cu concentrations (ranging from 1.54 to 1.89 µg/g d. m. and falling into the interval of deficiency) were determined in Juniperus communis scrubs (F) and transition mires and quaking bogs (D). Compared to other habitat types, transition mires and quaking bogs (D) and xero-thermophile fringes (E) had the lowest concentrations of Mn (ranging from 56.7 to 42.2 µg/g d. m.) that were still within the interval of concentrations required for the normal growth (20 to 300 µg/g d. m.) [69]. Fe chlorosis often occurs when the concentration of Fe is decreased [70] or if the concentration of Mn exceeds that of Fe by twice or more [71]. Thus, Fe should always be greater than Mn in the environment to avoid the blockage of Fe. In our sites, the situation is not favorable in respect of the Fe and Mn ratio due to the low range of Fe concentrations (20.2 to 115 µg/g d. m.) and the high range of Mn concentrations (30.2 to 939.2 µg/g d. m.). Elevated Cu concentration can also lead to Fe deficiency [62], but this was not the case in our study as Cu concentrations in J. communis were within the range of either deficiency (1.10 to 7.46 µg/g d. m.) or normal growth (2 to 3 µg/g d. m.) [48].
Compared to other habitats, the concentration of elements in the needles of J. communis in subcontinental moss Scots pine forests (G) were high for N, P, Mg, and Mn, moderate for K, Zn, and Cd, and low for Fe, Na, Cr, and Pb. Subcontinental moss Scots pine forests (G) are distinguished by having optimal conditions for J. communis, which forms forest undergrowth below the crown of Scots pine. In this habitat, significantly higher concentrations of K (3266 µg/g d. m.) and P (1765 µg/g d. m.) were determined when compared to other habitats. The lowest concentration of Pb (0.186 µg/g d. m.) was found in this habitat, although it did not differ significantly from the xero-thermophile fringes (E). The concentration of Pb did not fall between the interval of concentrations known as toxic (5 to 10 µg/g d. m.) [54]. The comparison of elemental concentrations in the needles of J. communis between xero-thermophile fringes (E) and other habitats revealed that in xero-thermophile fringes the concentrations of Mg, Cu, and Al were high, concentrations of P, Ni, Na, and Cr were moderate, and concentrations of Mn and Fe were low. The highest Mg concentrations (613 µg/g d. m.) were found in both subcontinental moss Scots pine forests (G) and xero-thermophile fringes (E), representing the levels required for normal growth.
In most cases, needle concentrations of N, P, K, and Mg were positively interrelated, but negatively related to Ca (Figure 3). Positive correlations were found between needle concentrations of Pb, Cr, and Ni. In some cases, Zn and Cu positively correlated with macroelements. Increased concentrations of Al corresponded to increased concentrations of Cu and Zn and to decreased concentrations of Al, Fe, Mn, Zn, Pb, Cr, and Cd. In case of significant correlations between Pb, Cr, and Cd, the higher concentration of one heavy metal corresponded to the higher concentration of the other.
Discriminating between the populations (Figure 4), the most important variables for the PC1 were concentrations of Cd, Al, Cr, and Fe and the most important for the PC2 were concentrations of P, N, Mg, Ca, Cu, and K. Discriminating between the habitats ( Figure 5), important variables for the PC1 were concentrations of Fe and Cd, while concentrations of N and P were important for the PC2. Principal component analyses using concentrations of 15 elements as variables for the discrimination between populations or habitats allowed authors to distinguishing the F and B habitats from the E habitat (PC1) and F and D habitats from the G habitat (PC2).
In parallel to ionomic studies of J. communis, genetic diversity at inter-simple sequence repeat (ISSR) loci was analyzed [39] and coverage of J. communis was estimated. Molecular diversity (percentage of polymorphic ISSR loci) of J. communis was lowest for transition mires and quaking bogs (D, 42.9% as a mean) and highest for Juniperus communis scrubs (F, 68.2%). Molecular diversity was intermediate for subcontinental moss Scots pine forests (G, 43.9% as a mean), for coastal brown dunes covered with natural Scots pine forests (B, 48.0 %), and for xero-thermophile fringes (E, 48.5%) [72]. Coverage by J. communis also depended on the habitat type, the smallest being for transition mires and quaking bogs (D, 5 % as a mean) and the highest for Juniperus communis scrubs (F, 60%). It was intermediate for subcontinental moss Scots pine forests (G, 13% as a mean), for coastal brown dunes covered with natural Scots pine forests (B, 15%), and for xero-thermophile fringes (E, 36%) [72]. As such, the lowest polymorphism at ISSR loci and the lowest coverage by J. communis in transition mires and quaking bogs corresponded the worst nutritional status in respect to needle concentrations of N, P, Mg and Cu. The highest polymorphism and the highest coverage by J. communis in Juniperus communis scrubs (F) corresponded to the highest needle concentration of K and deficiency in P, Mg, Zn, and Cu.
For various plant species, including junipers, Ellenberg indicator values (EIV) of neighboring herbaceous plant species are widely used to evaluate environment in the sites without the need to perform physico-chemical measurements [73][74][75][76][77]. During evaluation of J. communis environments by EIV, the soil richness in nutrients (EIV-N) [35] showed low fertility for all habitats; it was lower than the middle value (5) and ranged in the interval of 2.7 to 4.4. Soil reaction (EIV-R) ranged in the wider interval of 2.7 to 7.4 [72]. Our soil acidity values corresponded well with the findings of chemical analyses performed by other researchers [78]. According to pH, the most calcareous soil (EIV-R-7. Juniperus communis belongs to the list of plant taxa most frequently mentioned in the records of Lithuanian, Latvian, and Estonian folk medicine. Juniper was grown as a houseplant and used to treat respiratory and skin diseases. It was also applied as Sauna whisk. Juniper tea was used against bad breath, headache, hypersalivation, abdominal pain, chest pain, scabies, bacterial infection, tuberculosis, etc. [34,[79][80][81]. This species is included into Estonian, Latvian, and Lithuanian national pharmacopoeias [82]. In order to clarify the best conditions for collecting medicinal raw material of juniper depending on the condition of the plant and environmental factors, intensive qualitative and quantitative analysis of essential oils has been conducted in Lithuania and other Baltic countries [22,23,78,79,83,84]. Our elemental analysis revealed that the ionome of juniper growing in distinct habitats is different, therefore junipers collected in a single habitat would be the most suitable for a homogeneous medicinal raw material. According to the nutritional status of the needles, junipers growing in the subcontinental moss Scots pine forests (G) would be the most suitable for this purpose.

Study Sites and Sampling Material
For the study, 14 sites of J. communis representing five different habitats and covering most of Lithuania's territory was selected ( Figure 6). Following classification by Davies et al. (2004), habitat types were named as follows: (1)   whisk. Juniper tea was used against bad breath, headache, hypersalivation, abdominal pain, chest pain, scabies, bacterial infection, tuberculosis, etc. [34,[79][80][81]. This species is included into Estonian, Latvian, and Lithuanian national pharmacopoeias [82]. In order to clarify the best conditions for collecting medicinal raw material of juniper depending on the condition of the plant and environmental factors, intensive qualitative and quantitative analysis of essential oils has been conducted in Lithuania and other Baltic countries [22,23,78,79,83,84]. Our elemental analysis revealed that the ionome of juniper growing in distinct habitats is different, therefore junipers collected in a single habitat would be the most suitable for a homogeneous medicinal raw material. According to the nutritional status of the needles, junipers growing in the subcontinental moss Scots pine forests (G) would be the most suitable for this purpose.

Study sites and sampling material
For the study, 14 sites of J. communis representing five different habitats and covering most of Lithuania's territory was selected ( Figure 6). Following classification by Davies et al. (2004), habitat types were named as follows: (1)   Geographic distances between sites ranged from 46 to 339 km. Two pairs of neighboring sites (3 D and 4 G, and 7 G and 8 D) located within 0.5 km distance of each other were included as they differed in habitat type. Geographical ranges between the study sites covered 56°07′ to 54°11′ latitude, 21°06′ to 26°30′ longitude and 25 to 187 m altitude [39].
Sampling was carried out during a 2-day period within the 2nd week of September 2013. At each site, shoots with current-year needles were harvested from the middle part of the crown. If a tree or a bush was higher than 2.5 m then shoots were taken at the height of 1.75 to 2.00 m. The middle of September was chosen for sampling because current-year needles finish their growth at this time. Current-year needles of J. communis were examined in many other ionomic studies [33,46,47]. Needle loss starts from the second or third year and differs depending on the site; therefore, mature current-year needles of J. Geographic distances between sites ranged from 46 to 339 km. Two pairs of neighboring sites (3 D and 4 G, and 7 G and 8 D) located within 0.5 km distance of each other were included as they differed in habitat type. Geographical ranges between the study sites covered 56 • 07 to 54 • 11 latitude, 21 • 06 to 26 • 30 longitude and 25 to 187 m altitude [39].
Sampling was carried out during a 2-day period within the 2nd week of September 2013. At each site, shoots with current-year needles were harvested from the middle part of the crown. If a tree or a bush was higher than 2.5 m then shoots were taken at the height of 1.75 to 2.00 m. The middle of September was chosen for sampling because current-year needles finish their growth at this time. Current-year needles of J. communis were examined in many other ionomic studies [33,46,47]. Needle loss starts from the second or third year and differs depending on the site; therefore, mature current-year needles of J. communis are most suitable for elemental analysis. Because of the varying opinions regarding the sexual dimorphism of and differences in the chemical composition between female and male individuals [33,44,47,85], samples were taken only from female plants to ensure that plant material was as homogenous as possible. Moreover, female seed cones are used for medical and economical purposes [30,31,79,86]. At each site, three independent batches of needles were collected from the same individuals used for molecular studies [39]. Shoots were placed in separate paper bags and dried at room temperature, then placed in a thermostat at 30 • C until a constant mass was attained. Any small fragments of the bark or buds of the previous year's shoots were carefully eliminated. Needles were milled into fine powder using a Retsch MM400 tungsten carbide grinder (Germany).

Analysis of Nitrogen and Macro-, Micro-and Non-Essential Elements
The Kjeldahl method [87] was used to determine nitrogen (N) in the current-year needles of J. communis. Milled samples were digested in a Semi-Automatic Digester Instrument, model DK-20S (VELP Scientifica, Italy). Ammonia distillation using the Kjeldahl standard method was performed in an Automatic Distillation and Titration System, model UDK 159 (VELP Scientifica, Italy) as described earlier [88]. Nitrogen concentration was measured as micrograms per gram in a dry mass (µg/g d. m.). The quality assurance of analysis was ensured using standard reference materials NIST 1515 and NIST 1575 and a certificated reference material CRM 125045.
To determine macro-, micro-, and non-essential elements in current-year needles of J. communis, milled samples were digested in an ETHOS One microwave digestion system with a high pressure segmented rotor SK10 (Milestone, Italy). Digested samples were analysed by atomic absorption spectrophotometer, model AA-6800, controlled by WizAArd 2.31 (Shimadzu, Japan) software. Calibration lines for each element were constructed using standard solutions (1000 µg/mL, Sharlau, Spain). Standard reference materials NIST 1575 and NIST 1547 were used for calibration quality assurance.
Phosphorus concentration of mineralized J. communis samples was determined spectrophotometrically [89]. The colorimetric analysis was carried out using an acidic molybdate solution and ascorbic acid. Measurements were performed at a wavelength of 627 nm with a dual-beam spectrophotometer UV-1800 (Shimadzu, Japan).

Comparison of Ionomic Data with Environmental Parameters
To relate ionomic data with other characteristics of J. communis, the genetic diversity of juniper populations was evaluated using ISSR markers [39]. For comparison of ionomic data with environmental parameters, undergrowth trees and shrubs, herbaceous species, and dwarf shrubs were recorded at each site following Braun-Blanquet [90] and the abundance of each species was evaluated using EIV [35] as described in detail previously [72,75,76].

Statistical Analysis
To compare different populations and population groups representing different habitats, descriptive statistics were calculated using R software package (v. 4.2.2) and R package "DescTools" (v. 0.99.42) [91,92]. To compare macro-, micro-, and non-essential elements concentrations between population groups, the Kruskal-Wallis test was used [93]. To relate macro-, micro-, and non-essential elements, Pearson rank correlations were calculated and a correlogram was constructed using R package "corrplot" (v. 090) [94]. Principal component analysis was carried out using macro-, micro-, and non-essential elements as variables using R software [92].

Conclusions
1. Of all elements analyzed in current year needles, the concentrations of N and P were the most important for the discrimination between J. communis habitats.
2. The worst nutritional status according to the concentrations of N, P, Mg, and Cu in the needles of J. communis in transition mires and quaking bogs corresponded to the lowest polymorphism at ISSR loci and the lowest coverage of this species.